Autofocus imaging for a microscope

ABSTRACT

The present invention relates to the field of digital pathology and in particular to whole slide scanners. Autofocus imaging can be performed by sampling a first number of pixels of a primary image sensor and sampling a second number of pixels of an autofocus image sensor, wherein the second number is between one quarter and three quarters of the first number. Thus, continuous autofocus for rapid light scanning may be provided based on an additional image sensor that is tilted with respect to the optical axis.

FIELD OF THE INVENTION

The present invention relates to the field of digital pathology,notably. In particular, the present invention relates to an autofocusimaging system for a microscope, a microscope comprising an autofocusimaging system, a method for autofocus imaging of a microscope, acomputer-readable medium and a program element.

BACKGROUND OF THE INVENTION

In digital pathology and particular in the case of whole slide scanning,specimens are sliced and imaged for analysis purposes as well asteaching purposes. Line sensors may be used for scanning a whole tissueslide. These slide scanners may perform a continuous mechanicalscanning, thereby reducing stitching problems and allowing for the useof so-called time delay integration (TDI) line sensors in order toaccommodate for low brightness of the illumination.

For focusing focus maps may be used. Before the actual scanning theoptimum focus position is determined at a number of positions on theslide. This results in a “focus map”. This procedure may be necessarybecause the axial position of the tissue layer may vary with severalmicrometers across the slide, as may be seen in FIG. 1. The variation ofthe tissue layer may thus be more than the focal depth of the microscopeobjective. During scanning the focus position of the objection is set ona trajectory that interpolates between the measured optimum focussettings on the selected measurement locations. This procedure may beboth prone to errors and be also time-consuming, thereby limiting thethroughput of the system.

WO 2005/010495 A2 describes a system and a method for generating digitalimages of a microscope slide, the microscope comprising a main cameraand a focus camera which is tilted with respect to the optical axis.

SUMMARY OF THE INVENTION

However, the performance of the autofocus function may be insufficient.

It may be desirable to have an autofocus imaging system with improvedperformance.

According to a first aspect of the invention an autofocus imaging systemfor a microscope is provided, which comprises a primary image sensor andan autofocus image sensor. The primary image sensor is adapted foracquiring primary image data of an object of interest, such as a tissueslide. The autofocus image sensor is adapted for acquiring autofocusimage data of an oblique section of the object of interest. The primaryimage sensor is further adapted for sampling a first number of pixelsper distance in object space and the autofocus image sensor is furtheradapted for sampling a second number of pixels per distance in objectspace, wherein the second number is between one quarter and threequarters of the first number.

In other words, the autofocus image sensor samples a smaller number ofpixels per distance in object space than the primary image sensor. Bysampling a smaller number of pixels, the computational load and also thesampling time may be reduced. Furthermore, by sampling not less than onequarter of the pixels which are sampled by the primary image sensor thequality of the autofocus sensor signal may be optimized.

According to an exemplary embodiment the second number is half of thefirst number. In other words, the autofocus image sensor samples halfthe numbers of pixels per distance in object space in the primary imagesensor.

The primary image sensor assembly may comprise one line sensor or maycomprise more than one line sensor, for example three or even more linesensors. Each line sensor may detect a different wavelength orwavelength range. For example, one line sensor may detect green light, asecond red light and a third line sensor may detect blue light (only).

According to another exemplary embodiment the autofocus image sensor istilted with respect to an optical axis of radiation from the object ofinterest towards the autofocus image sensor, e.g. tilted with respect toan optical axis of the primary image sensor. In this way the position ofthe tissue layer on the sensor is a measure for the amount of defocus.

According to another exemplary embodiment the autofocus image sensor isadapted for acquiring the autofocus image data at a light frequencyoutside the frequency of the visible spectrum.

According to another exemplary embodiment the autofocus imaging systemis adapted for dark field illumination of the autofocus image sensor.

In other words, the object of interest may be illuminated with a beamcomprising a set of directions of propagation, such that the angle ofthese directions of propagation is larger than the angle sub-tended bythe detection aperture of the autofocus imaging sensor. In this waylight reflected from various surfaces (air, cover slip, coverslip-tissue layer, tissue layer-slide, slide-air) may not end up at theautofocus image sensor. In fact, all low object spatial frequencies maybe blocked and only signal emanating from the tissue (which hassufficiently high spatial frequencies) may be detected at the autofocusimage sensor. This may improve the robustness and accuracy that theaxial position of the tissue layer can be measured.

According to a second aspect of the invention a microscope comprising anabove and below described imaging system is provided.

According to an exemplary embodiment of the invention, the microscope isadapted as a slide scanner for digital pathology.

According to another aspect of the invention a method for autofocusimaging of a microscope is provided, in which primary image data of anobject of interest is acquired by a primary image sensor, autofocusimage data of an oblique section of the object of interest is acquiredby an autofocus image sensor, a first number of pixels per distance inobject space are sampled, the first number of pixels being pixels of theprimary image sensor, and a second number of pixels per distance inobject space is sampled, the second number of pixels being pixels of theautofocus image sensor. The second number is between one quarter andthree quarters of the first number.

According to another aspect of the invention a computer-readable mediumis provided, in which a computer program for autofocus imaging of amicroscope is stored which, when executed by a processor of amicroscope, causes the processor to carry out the above and/or belowdescribed method steps.

Furthermore, according to another aspect of the invention, a programelement for autofocus imaging of a microscope is provided, which, whenbeing executed by a processor of a microscope, causes the processor tocarry out the above and/or below described method steps.

A computer-readable medium may be a floppy disk, a hard disk, a CD, aDVD, an USB (Universal Serial Bus) storage device, a RAM (Random AccessMemory), a ROM (Read Only Memory) and an EPROM (Erasable ProgrammableRead Only Memory). A computer-readable medium may also be a datacommunication network, for example the Internet, which allowsdownloading a program code.

It may be seen as a gist of an exemplary embodiment of the presentinvention, that the autofocus imaging sensor, which may be atwo-dimensional sensor, samples a smaller number of pixels per distancein object space and the primary sensor, which may be a line sensor orwhich may comprise more than one line sensors. For example, theautofocus sensor samples half the number of pixels than the primarysensor.

These and other aspects of the invention will be apparent from andelucidated with reference to the embodiments described hereinafter.

Exemplary embodiments of the present invention will now be described inthe following, with respect to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-section of a tissue slide assembly.

FIG. 2 shows a tilted autofocus image sensor.

FIG. 3 shows the effect of defocus on MTF.

FIG. 4 shows the ratio of defocus MTF to zero defocus MTF.

FIG. 5 shows a microscope with an autofocus imaging system according toan exemplary embodiment of the invention.

FIG. 6 shows a microscope with an autofocus imaging system according toanother exemplary embodiment of the invention.

FIG. 7 shows a microscope with an autofocus imaging system according toanother exemplary embodiment of the invention.

FIG. 8 shows a microscope system according to an exemplary embodiment ofthe invention.

FIG. 9 shows a flow-chart of a method according to an exemplaryembodiment of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

The illustration in the drawings is schematically. In differentdrawings, similar or identical elements are provided with the samereference numerals.

In the following, the character prime (′) associated to a symbol willmean that the image space is considered (e.g. sensor reference) while asymbol without prime character will mean that the object space isconsidered (typically the sample reference). For example, when the angleBeta prime (β′) will be used in this description, a rotation in imagespace, and, as will be described more specifically, a rotation of thephysical sensor, will be indicated. Also, an angle Beta (β withoutprima) will indicate a rotation in object space, and as will bedescribed more specifically a rotation of an oblique cross section ofthe sample that is imaged by the autofocus sensor.

FIG. 1 shows a schematic cross-section of a tissue slide assembly,comprising a microscope slide 1, having a typical thickness of 1 mm, acover slip 2, with a typical thickness of 0.17 mm, a mounting medium 3for fixing and sealing off a tissue layer 4. The tissue layer istypically around 5 μm thick, the mounting layer includes the tissuelayer and is typically 10-15 μm thick. The mounting medium may beapplied to the slide with tissue layer in liquid form before a coverslip is attached to the slide, subsequently the mounting liquidsolidifies, thus mechanically fixing the tissue layer and sealing it offfrom the outside environment in order to provide stability againstdeterioration. The axial position of the tissue layer may vary withinseveral μm across the slide.

For providing an optimum resolution during scanning the focus may haveto be adjusted continuously, since the axial position of the tissuelayer varies.

An alternative for the use of the “focus map”-method is the use of acontinuous autofocus system, i.e. an additional system that continuouslymeasures the optimum focus position and adapts the axial position of theobjective lens during the actual scan for acquiring the digital image.The autofocus system may be based on optimizing the contrast in theobtained image. A variety of matrix may be used for contrastoptimization. However, the sine of the focus error (above or belowfocus) can not be determined in this manner, i.e. the focus error signalis not polar. This may be disadvantageous for a continuous autofocussystem that needs permanent updates on the optimum focus setting.

The autofocus system may use the line reflected at a reference surfaceat or near the object plane, such as in optical disks. However, adrawback of this method when applied to tissue slides may be that therelevant interface (between microscope slide and tissue layer andbetween tissue layer and cover slip) may have a low reflectance and thatthe reflection signal is distorted by scattering arising from the nearbytissue layer, thus comprising robustness.

A good alternative is the use of an additional sensor that is tiltedwith respect to the optical axis. This autofocus image sensor makes animage of an oblique section of the object, as depicted in FIG. 2. Thissection may cut through the tissue layer at some point depending on theaxial position of the tissue layer or relative to the focal plane of theobjective lens. In this way the position of the tissue layer on thesensor is a measure for the amount of defocus. For more details on theseaspects, the reader may refer to the European patent application No09306350.

As can be seen from FIG. 2 the tilted autofocus image sensor makes animage of an oblique cross-section 5 of the tissue slide assembly. Thetilt is in the scanning direction 6. The sensor has N_(x) pixels andsamples the object in the scan direction with Δx per pixel and in theaxial direction with Δz per pixel.

For example, the autofocus imaging system operates using wavelengthsoutside the visible spectrum so as not to spoil the white light imagingof the tissue layer. For example, the autofocus system operates usingwavelengths on the infrared side of the visible spectrum, becauseultraviolet radiation may damage the tissue and may require morecomplicated and/or expensive optical components than infrared radiation.

In an exemplary embodiment, the additional autofocus image may beprovided by using a so-called dark field illumination. Hereby, thesample is illuminated with a beam comprising a set of directions ofpropagation, as already described above.

A problem may arise if the tilted autofocus sensor is combined with atime delay integration (TDI) line sensor (primary image sensor) for highthroughput imaging. Such a TDI-line sensor records each object pixel Ltimes, where the number of stages L can be typically up to 128. This hasthe effect that the total integration time, and hence signal level,increases by a factor L compared to a conventional single line sensor.This is used to increase the scanning speed of the system.

A reasonable starting point in the design of such a system may entailhaving a resolution R_(af) of the autofocus sensor approximately equalto the resolution R_(im) of the (TDI-based) image sensor in order to beable to test the same level of sharpness in the image. The novel insightof the inventors is that this implies a problem with signal level on theautofocus image sensor as will be apparent from the followingconsiderations. Taking a linear scan speed v the line rate of the imagesensor is:

$\begin{matrix}{\frac{1}{T_{im}} = \frac{2\; v}{R_{im}}} & (1.1)\end{matrix}$

(NB: pixel size=half the resolution) making the total integration timeLT_(im). In order to prevent motion blur the autofocus sensor must havea shutter such that the collection time is:

$\begin{matrix}{T_{af} = \frac{R_{af}}{2\; v}} & (1.2)\end{matrix}$

The beam after the objective lens is split in two parts by a beamsplitter, a fraction η is directed towards the autofocus sensor, and afraction 1-η towards the image sensor. If the slide is illuminated withan intensity B (incident power per area), then the signal level at theimage sensor and at the autofocus image sensor are given by:

$\begin{matrix}{{I_{im} = {{\eta_{im}( {1 - \eta} )}L\frac{B}{v}( \frac{R_{im}}{2} )^{3}}}{I_{af} = {\eta_{af}\eta\frac{B}{v}( \frac{R_{af}}{2} )^{3}}}} & (1.3)\end{matrix}$

where η_(im) is the image sensor (quantum) efficiency and η_(af) is theautofocus sensor (quantum) efficiency. These sensor efficiencies may beassumed to be approximately equal. The ratio of the two is:

$\begin{matrix}{\frac{I_{af}}{I_{im}} = {\frac{\eta_{af}}{\eta_{im}}\frac{\eta}{( {1 - \eta} )L}( \frac{R_{af}}{R_{im}} )^{3}}} & (1.4)\end{matrix}$

If L₀=(1−η)L is the number of stages that would be needed if noautofocus sensor was used and taking R_(af)≈R_(im) and η_(af)≈η_(im) itfollows that:

$\begin{matrix}{\frac{I_{af}}{I_{im}} \approx \frac{\eta}{L_{0}} ⪡ 1} & (1.5)\end{matrix}$

Clearly, the signal level at the autofocus sensor is much smaller thanthe signal level at the image sensor. As a consequence, the autofocussensor signal will be relatively noisy, which compromises the accuracyof the focus error signal.

There may be a significant redundancy in the resolution requirements onthe autofocus sensor compared to the resolution requirements on theimage sensor. This insight follows from the study of the effect ofdefocus on the so-called Modulation Transfer Function (MTF), which isthe ratio of the modulation in the image of a periodic object and themodulation in the object itself as a function of spatial frequency (theinverse of the period p). The MTF as a function of defocus for thesimplified 1D-case with equal condenser and objective NA is given by:

$\begin{matrix}{{MTF} = {\sin\;{c( {2{\pi\beta}\;{q( {2 - q} )}} )}( {1 - \frac{q}{2}} )}} & (1.6)\end{matrix}$

with sinc(x)=sin(x)/x, q=λ/pNA is the normalized spatial frequency, andβ—ΔzNA²/2nλ is a defocus parameter. FIG. 3 shows the MTF for the nominalin-focus situation 301 and for a case with defocus 302. The x-axis 303depicts the normalized spatial frequency and the y-axis 304 depicts theMTF values.

FIG. 4 shows the ratio 401 of the two MTF-functions 301, 302. The y-axis403 depicts the MTF ratio; a minimum 402 can be observed the x valueequal to 1. Both MTF-functions show a cut-off at 2NA/λ, (this is theso-called ‘diffraction limit’), which is the ultimate resolution limitfor a conventional microscope. The ratio of the two MTF-functions showsa dip for the middle spatial frequencies. From this analysis we mayconclude that:

-   -   The resolution of the primary image sensor is preferably        determined by the so-called Nyquist-criterion for the 2NA/λ,        spatial frequency cut-off to R_(im)=λ/2NA (so pixel size        M_(im)λ/4NA, with M_(im) the magnification from object to image        sensor).    -   The resolution of the autofocus sensor is preferably determined        by the maximum in defocus sensitivity to half the spatial        frequency cut-off to R_(af)=λ/NA (so pixel size M_(af)λ/2NA,        with M_(af) the magnification from object to autofocus sensor).

According to an exemplary embodiment the autofocus image sensor sampling(pixels/m in object space) is selected ¾ to ¼ or, e.g., at least afactor two smaller than the image sensor sampling. This gives a goodcompromise between defocus sensitivity and autofocus to image signalratio. Preferably, the beam splitter fraction η is adapted such that theautofocus sensor signal is sufficiently high compared to the imagesensor signal. Preferably, the parameter settings are such that theTDI-based line sensor has sufficient redundancy to maintain asufficiently high image sensor signal, i.e. η>1−L₀/L_(max), whereL_(max) is the maximum number of TDI-stages.

This is different from the implementation of a secondary cameraautofocus method based on the addition of a dedicated image sensor forautofocus, which is not tilted with respect to the plane in the objectthat is being imaged, and where the difference in resolution(specifically a lower resolution of the autofocus sensor) serves thesole purpose of increasing the speed of the autofocus sensor withrespect to the primary image capturing sensor. Also the reduction inpixel count is described for several embodiments as a factor of at least3, and a factor of at least 10. As is seen in the minimum in the bottomgraph of FIG. 3, the inventors specifically found an optimum at areduction in the resolution of exactly 2. Although a practical range fora second embodiment would be a range between 4/3 and a factor 4.

The depth range Δz_(tot) of the autofocus system must be sufficientlylarge for realistic settings of other parameters. The autofocus imagesensor has N_(x) pixels in the scan direction, with pixel size b. Thesensor is tilted over an angle β′ so that the lateral and axial samplingis given by:Δx′=b cos β′Δz′=b sin β′

The lateral and axial sampling at the object (the tissue slide) is givenby:Δx=Δx′/MΔz=nΔz′/M ²

where M is the magnification and n the refractive index of the object.The axial sampling at the object now follows as:

${\Delta\; z} = {\frac{n\;\Delta\; z^{\prime}}{( {\Delta\;{x^{\prime}/\Delta}\; x} )^{2}} = {\frac{\sin\;\beta^{\prime}}{\cos^{2}\beta^{\prime}}\frac{n\;\Delta\; x^{2}}{b}}}$

As there are N_(x) pixels the total depth range is:

${\Delta\; z_{tot}} = {{N_{x}\Delta\; z} = {\frac{\sin\;\beta^{\prime}}{\cos^{2}\beta^{\prime}}\frac{N_{x}n\;\Delta\; x^{2}}{b}}}$

Table 1 shows an example of parameter settings according to theinvention. In this example the autofocus resolution is 2×0.9 μm, whereasthe image resolution is preferably about 2×0.25 μm (taking a 20×/NA0.75microscope objective).

As a non limitative example, FIG. 5 shows part of a microscope and inparticular the imaging branch of the light path. An embodiment forepi-mode dark field illumination is shown in FIG. 6.

The light passing through the slide 1 and the cover slip 2 (and tissuelayer 4, not shown) is captured by the objective lens 20 with the backaperture 21, wherein the unscattered beams are blocked. A coloursplitter 22 splits off the white light which is imaged by a tube lens 23onto the image sensor arrangement, which may comprise a first, a secondand a third primary image sensor 24, 32, 33, which may be adapted in theform of line sensors, 24 for generating the digital tissue image. Theinfrared light is imaged by a second tube lens 25 onto the autofocusimage sensor 26, which is tilted with respect to the optical axis 31 ofradiation from the object of interest towards the autofocus image sensor26. In the context of this disclosure “tilted with respect to theoptical axis of the primary image sensor” means that the radiation fromthe object of interest which impinges on the autofocus image sensor doesnot impinge on the autofocus image sensor perpendicularly. However, theradiation which travels from the object of interest towards the primaryimage sensor may impinge perpendicularly on the primary image sensor,although this is not required already described herein above. Raysscattered by the tissue can pass through the aperture 21 and are imagedonto the autofocus image sensor 26.

FIG. 6 shows an optical layout for epi-mode dark field illumination of amicroscope with an autofocus imaging system 500 having a laser diode 14,the illumination being integrated with the imaging branch. Two crossedgratings 15 are arranged after the laser diode 14 for generatingdiffraction orders, for example a 0th diffraction order S′₀, a +1^(st)order S′₊₁ and a −1^(st) order S′⁻¹. Still further, a field stop 16 isarranged close to the gratings 15 for limiting the width of the darkfield illumination beams, and a collimator lens 17 the collimates thelight from the laser diode 14.

A polarizing beam splitter 28 is provided to split the beam after it haspassed the collimator lens 17. Furthermore, the microscope comprisesquarter-wave plate 29. Both elements 28 and 29 take care of directingthe beam originating from the laser towards the objective lens anderecting the scattered light originating from the tissue towards theautofocus image sensor.

FIG. 7 shows an optical layout for multi-spot illumination of amicroscope 500, which illumination is integrated with the imagingbranch. The lens 17 collimates the beam which is incident on a spotgenerator for generating an array of spots 30. By tilting the wholeassembly, the spot array can be tilted so that the resulting incidentspot array and the slide is tilted as well. The spot generator 29generates an array of low-NA beams, which can pass the beam splitter 27without introducing significant aberrations.

In the embodiment of FIG. 7 an array of spots is used to illuminate theoblique section 5 that is imaged by the autofocus image sensor. Thespots that are focused on the tissue may experience time-dependentscattering as the absorption and refractive index of the region intowhich the spot is focused changes with scanning. By examining thetime-dependence of the spots imaged on the autofocus image sensor theaxial position of a tissue layer may be located. Namely, close to focusthe high resolution information is visible, away from focus this isblurred. As a consequence, the signal variations on a comparativelysmall time scale may be maximum when the tissue layer coincides with thefocal plane.

FIG. 8 shows a microscope system 802 comprising a microscope with anautofocus imaging system 500 connected to a processor or processing unit800 which is connected to a user interface 801, such as a computer.

FIG. 9 shows a flow-chart of a method according to an exemplaryembodiment. In step 901 primary and secondary, i.e. autofocus image dataof an object of interest are acquired by a primary image sensor and anautofocus image sensor, respectively. In step 902 the pixels of theprimary image sensor are sampled. In step 903 (which can be before,after or at the same time as step 902) a certain number of pixels perdistance in object space of the autofocus image sensor is sampled. Thisnumber is smaller than the sampled number of pixels of the primary imagesensor. Then, in step 904, the focus of the microscope is adjusted basedon the sampling.

Thus, the focus of the primary image sensor may be adjustedautomatically. In another embodiment of the invention, the principles ofthe present invention may be advantageously applied to a sensor whichthe applicant of the present invention has already proposed underEuropean patent application N°09306350, and which is hereby incorporatedby reference.

As a result, according to this embodiment the primary image sensor andthe autofocus image sensor may share a same sensing area. In otherwords, the primary image sensor and the autofocus image sensor maytogether form a unique sensor with a sensing area (typically formed ofpixels) that is both used for autofocus and for image acquisition.

According to this embodiment, the larger autofocus pixels may be eitheractual physical pixels located next to or intermixed in the array orarrays of primary image pixels, or the autofocus pixels may be virtualpixels obtained by combining two or more of the primary image pixelsinto a larger virtual autofocus pixel. Such a combination may be done onthe sensor itself, or in a separate processing unit.

The described autofocus system finds application in digital pathologyand other fields of rapid micro scanning

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive; theinvention is not limited to the disclosed embodiments. Other variationsto the disclosed embodiments can be understood and effected by thoseskilled in the art and practising the claimed invention, from a study ofthe drawings, the disclosure, and the appended claims. In the claims,the word “comprising” does not exclude other elements or steps, and theindefinite article “a” or “an” does not exclude a plurality. The merefact that certain measures are recited in mutually different dependentclaims does not indicate that a combination of these measures cannot beused to advantage. Any reference signs in the claims should not beconstrued as limiting the scope.

TABLE 1 Example of parameter settings. sensor pixel size (μm) 10.0 givennumbers #pixels x (scan) direction 640 #pixels y direction 480 sensortilt angle (deg) 12.0 tube lens focal length (mm) 100.0 objective lensmagnification 20 reference tube focal length (mm) 180 refractive indexslide 1.50 lateral magnification 11.1 calculated numbers axialmagnification 82.3 sampling x (scan) direction (μm) 0.88 sampling ydirection (μm) 0.90 sampling z (axial) direction (μm) 0.025 fieldx-direction (mm) 0.563 field y-direction (mm) 0.432 axial (z) range (μm)16.2

LIST OF REFERENCE SIGNS

1 microscope slide

2 cover slip

3 mounting medium

4 tissue layer

5 oblique cross-section

6 scanning direction

14 laser diode

15 two crossed gratings

16 field stop

17 collimator lens

18 stop for blocking Oth order light rays

20 objective lenses

21 back aperture

22 colour splitter

23 tube lens

24 first primary image sensor

25 tube lens

26 autofocus image sensor

28 beam splitter

29 quarter-wave plate

31 optical axis

32 second primary image sensor

33 third primary image sensor

301 MTF for nominal in-focus situation

302 MTF for defocus situation

303 x-axis (normalized spatial frequency)

304 y-axis (MTF)

401 ratio of the MTF functions 301, 302

402 minimum

403 y-axis (ratio of the MTF functions 301, 302)

500 autofocus imaging system

800 processor

801 user interface

802 microscope system

901 method step

902 method step

903 method step

904 method step

The invention claimed is:
 1. Autofocus imaging system for a microscope,the autofocus imaging system comprising: a primary image sensorarrangement comprising a primary image sensor (24) for acquiring primaryimage data of an object of interest (4); an autofocus image sensor (26)for acquiring autofocus image data of an oblique section of the objectof interest (4); wherein the primary image sensor (24) is configured forsampling a first number of pixels per distance in object space; whereinthe autofocus image sensor (26) is configured for sampling a secondnumber of pixels per distance in object space; wherein the second numberis between one quarter and three quarters of the first number.
 2. Theautofocus imaging system of claim 1, wherein the second number is halfof the first number.
 3. The autofocus imaging system of claim 1, whereina resolution of the primary image sensor is wavelength (λ)/(2*numericalaperture (NA)); and wherein a resolution of the autofocus image sensoris λ/(NA).
 4. The autofocus imaging system of claim 1, wherein theautofocus image sensor (26) is tilted with respect to an optical axis(31) of the primary image sensor (24).
 5. The autofocus imaging systemof claim 1, wherein the autofocus image sensor (26) is tilted in a scandirection (6) of the autofocus imaging system.
 6. The autofocus imagingsystem of claim 1, further comprising: a beam splitter (22) forsplitting a beam from the object into a first beam towards the primaryimage sensor and a second beam towards the autofocus image sensor;wherein the a fraction between an intensity of the second beam and anintensity of the first beam is bigger than (1−L₀/L_(max)), wherein L₀ isa number of stages needed to focus with no autofocus sensor and L_(max)is a maximum number of Time Delay Integration stages of the primaryimage sensor.
 7. The autofocus imaging system of claim 1, wherein theautofocus image sensor (24) is configured for acquiring the autofocusimage data at a light frequency outside the frequency of the visiblespectrum.
 8. The autofocus imaging system of claim 1, wherein theautofocus imaging system (500) is configured for dark field illuminationof the autofocus image sensor.
 9. The autofocus imaging system of claim1, wherein the primary image sensor arrangement further comprises asecond primary image sensor (32) and a third primary image sensor (33);wherein each of the primary image sensors of the primary image sensorarrangement is configured for detecting light of a different wavelength.10. The autofocus imaging system of claim 1, wherein the primary imagesensor (24) is a line sensor, and wherein the autofocus image sensor(24) is a two-dimensional sensor.
 11. The autofocus imaging system ofclaim 1, wherein the primary image sensor and the autofocus image sensorshare a same sensing area.
 12. A microscope (802) comprising anautofocus imaging system (500) of claim
 1. 13. A method for autofocusimaging of a microscope (802), the method comprising the followingsteps: acquiring primary image data of an object of interest by aprimary image sensor (24) of a primary image sensor arrangement;acquiring autofocus image data of an oblique section of the object ofinterest by an autofocus image sensor (26); sampling a first number ofpixels per distance in object space, the first number of pixels beingpixels of the primary image sensor (24); sampling a second number ofpixels per distance in object space, the second number of pixels beingpixels of the autofocus image sensor (26); wherein the second number isbetween one quarter and three quarters of the first number.
 14. Anon-volatile computer-readable storage medium, in which a computerprogram for autofocus imaging of a microscope is stored which, whenexecuted by a processor (800) of the microscope (802), causes theprocessor (800) to carry out the steps of: acquiring primary image dataof an object of interest from a primary image sensor (24) of a primaryimage sensor arrangement; acquiring autofocus image data of an obliquesection of the object of interest from an autofocus image sensor (26);sampling a first number of pixels per distance in object space, thefirst number of pixels being pixels of the primary image sensor (24);sampling a second number of pixels per distance in object space, thesecond number of pixels being pixels of the autofocus image sensor (26);wherein the second number is between one quarter and three quarters ofthe first number.
 15. A non-volatile computer readable program mediumhaving encoded thereon a program element for autofocus imaging of amicroscope, which, when being executed by a processor (800) of themicroscope (802), causes the processor to carry out the steps of:acquiring primary image data of an object of interest from a primaryimage sensor (24) of a primary image sensor arrangement; acquiringautofocus image data of an oblique section of the object of interestfrom an autofocus image sensor (26); sampling a first number of pixelsper distance in object space, the first number of pixels being pixels ofthe primary image sensor (24); sampling a second number of pixels perdistance in object space, the second number of pixels being pixels ofthe autofocus image sensor (26); wherein the second number is betweenone quarter and three quarters of the first number.